This renewal application proposes continuation of current studies of proteins that regulate the blood coagulation pathways and control thrombosis in a laboratory devoted to studies of molecular mechanisms responsible for thrombosis and hemostasis. Research will focus on the protein S(PS)-dependent regulation of the intrinsic coagulation pathway and on the regulation of PS activity by C4b-binding protein (C4BP). Since hereditary PS deficiency is associated with thrombotic disease, PS is an important physiologic antithrombotic factor. A major goal will be the elucidation of the mechanism of anticoagulant activity of PS. Structure-function relationships that underlie the expression of anticoagulant activity of PS will be defined. It is hypothesized that the anticoagulant activity of PS depends on its binding to activated protein C (APC) and to Factors Va and VIIIA, as demonstrated in our preliminary results. Since free PS is anticoagulant whereas PS bound to C4BP is not, the structure-function relationships responsible for the association of PS with C4BP will be studied. The hypothesis is that PS binds to the recently discovered beta chain of C4BP. Studies will take advantage of our preliminary results showing that a 115,000 MW heterodimer derived from C4BP containing one alpha and one beta chain binds PS. Work will include the specific aims of identifying molecular regions and sequences responsible for the binding of PS to APC, of PS to Factors Va and VIIIA, and of PS to C4BP. These interactions will be studied using purified proteins, polypeptide fragments, antibodies and synthetic peptides representing specific sequences of these proteins. Competitions between these various ligands for binding to PS will be studied to assess the similarity or identity of binding sites. Covalently crosslinked heterodimer complexes of APC:PS will be prepared and their anticoagulant properties characterized. The mechanism of action of PS will be studied in kinetic analyses of the influence of PS on the competitions between Factors Xa or IXa and APC in the inactivation of Factors VIIIA and Va, respectively. The anticoagulant properties of PS bound to the 115,000 KW alpha-beta heterodimer derived from C4BP will be characterized to understand the mechanism of inhibition of PS activity by C4BP. These studies will provide new knowledge about natural anticoagulant mechanisms and will be potentially useful for developing new approaches to regulate thrombosis.